The C-terminal region of the movement protein of Cowpea mosaic virus is involved in binding to the large but not to the small coat protein

Carvalho, Claudine M.; Wellink, J.; Ribeiro, S. G.; Goldbach, Rob; Lent, J.W.M. van

doi:10.1099/vir.0.19101-0

Cited by 50 publications

(29 citation statements)

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“…The MP-CP interactions necessary for virus local movement have already been described for a number of positive-strand RNA viruses (18). Evidence for direct binding between CP and MP both in vitro (Maize streak virus and Cowpea mosaic virus [CPMV]) and in vivo (Maize streak virus) has been reported (6,21). Moreover, it has been shown that CPMV virions are transported through tubules formed with CPMV MP via MP-CP interactions and deposited in the adjacent cell (37).…”

Section: Discussionmentioning

confidence: 96%

Identification of a Movement Protein of Rice Yellow Stunt Rhabdovirus

Huang

Geng

Ying

et al. 2005

J Virol

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Section: Discussionmentioning

confidence: 96%

Identification of a Movement Protein of Rice Yellow Stunt Rhabdovirus

Huang

Geng

Ying

et al. 2005

J Virol

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“…Furthermore, the observation that MP-YFP was immobile within tubules suggested that tubules consisted of a stable MP multimer. Previously, interaction between MP subunits has been shown to occur in vitro using a blot overlay assay (Carvalho et al, 2003), and .…”

Section: Discussionmentioning

confidence: 99%

“…Electron microscopy analysis revealed that these tubules were morphologically indistinguishable from tubules made by non-fused MP, except that GFP, which was fused to the C terminus of MP and thus present inside the tubule (Carvalho et al, 2003), prevented the incorporation of virus particles (Gopinath et al, 2003). MP-GFP also accumulated in peripheral punctate spots in protoplasts & Palukaitis, 1999; Heinlein et al, 1998;Huang et al, 2000; Satoh et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

Studies on the origin and structure of tubules made by the movement protein of Cowpea mosaic virus

Pouwels¹,

Velden²,

Willemse³

et al. 2004

Journal of General Virology

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Cowpea mosaic virus (CPMV) moves from cell to cell by transporting virus particles via tubules formed through plasmodesmata by the movement protein (MP). On the surface of protoplasts, a fusion between the MP and the green fluorescent protein forms similar tubules and peripheral punctate spots. Here it was shown by time-lapse microscopy that tubules can grow out from a subset of these peripheral punctate spots, which are dynamic structures that seem anchored to the plasma membrane. Fluorescence resonance energy transfer experiments showed that MP subunits interacted within the tubule, where they were virtually immobile, confirming that tubules consist of a highly organized MP multimer. Fluorescence recovery after photobleaching experiments with protoplasts, transiently expressing fluorescent plasma membrane-associated proteins of different sizes, indicated that tubules made by CPMV MP do not interact directly with the surrounding plasma membrane. These experiments indicated an indirect interaction between the tubule and the surrounding plasma membrane, possibly via a host plasma membrane protein. INTRODUCTIONFor successful systemic infection, a plant virus must spread throughout the plant, a process that starts with transport from the initially infected cell to neighbouring uninfected cells (cell-to-cell movement) and is followed by transport through the phloem into roots and young developing leaves (systemic movement). Since plant viruses cannot pass through the rigid cell wall, they have evolved ways of exploiting plasmodesmata, the naturally occurring transport channels present between plant cells (Haywood et al., 2002). Normally, only small molecules are able to pass through plasmodesmata, but plant viruses encode one or more proteins, the so-called movement proteins (MPs), that modify the structure of plasmodesmata in such a way that viral transport is enabled. So far, two basic principles for cell-to-cell movement of plant viruses have been described: tubule-guided movement of virus particles and movement as ribonucleoprotein complexes (Lazarowitz & Beachy, 1999).Cowpea mosaic virus (CPMV), a positive-stranded, bipartite RNA virus belonging to the family Comoviridae, moves from cell to cell by transporting virus particles using tubular structures, which connect the infected cell to the neighbouring uninfected cell (Pouwels et al., 2002a). Immunogold labelling has shown that the CPMV MP is present in these tubular structures (van Lent et al., 1990). On the surface of CPMV-infected protoplasts, similar tubular structures are formed, which protrude up to 20 mm into the culture medium, are tightly surrounded by the plasma membrane and have the same ultrastructure as tubules in plant tissue (van Lent et al., 1991). Remarkably, tubules are also formed on protoplasts transiently expressing MP , showing that MP is the only viral protein required for tubule formation. So far, protoplasts have proved extremely useful as a model system for studying targeting and assembly of both wt and mutant MPs (Bertens et al., 20...

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“…To investigate potential rNTP-binding properties of CPMV MP, GTP-coupled agarose beads (Sigma), prewashed three times with binding buffer (24), were incubated for 60 min at 4°C with 5 g of purified wild-type MP (wtMP; prepared as described previously [3]). The beads were then pelleted by centrifugation (5 min at 18,000 ϫ g) and washed three times with binding buffer to remove unbound proteins.…”

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The Movement Protein of Cowpea Mosaic Virus Binds GTP and Single-Stranded Nucleic Acid In Vitro

Carvalho

Pouwels

Lent³

et al. 2004

J Virol

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The movement protein (MP) of Cowpea mosaic virus forms tubules in plasmodesmata to enable the transport of mature virions. Here it is shown that the MP is capable of specifically binding riboguanosine triphosphate and that mutational analysis suggests that GTP binding plays a role in the targeted transport of the MP. Furthermore, the MP is capable of binding both single-stranded RNA and single-stranded DNA in a nonsequence-specific manner, and the GTP-and RNA-binding sites do not overlap.Most plant virus genomes code for one or more movement proteins (MPs), which are required for viral cell-to-cell movement. Based on their primary structure, MPs can be divided into several superfamilies, one of which is the "30K" superfamily, related to the Tobacco mosaic virus (TMV) MP (20). Within this 30K superfamily, two basic mechanisms for cell-tocell movement have been proposed (18). TMV MP typifies one mechanism whereby the MP modifies plasmodesmata, allowing viral RNA-MP complexes to move from cell to cell. The other type of movement, best known from Cowpea mosaic virus (CPMV) MP, is the tubule-guided movement of mature virus particles through drastically modified plasmodesmata. Secondary-structure comparisons of MPs belonging to the 30K superfamily predicted a common central core (20). In this core, one aspartic acid, referred to as the D motif, is almost absolutely conserved (16, 21), but its function has so far remained unresolved. MPs of como-and tobamoviruses have been suggested to contain a Walker B-like ribonucleoside triphosphate (rNTP)-binding motif (4, 27), and some MPs of the 30K superfamily (for example, TMV MP) have been shown to bind GTP (19). However, the GTP-binding site has not been identified, and a function for GTP binding has not been described yet, although it has been suggested that GTP hydrolysis provides the energy needed for cell-to-cell transport (19).To investigate potential rNTP-binding properties of CPMV MP, GTP-coupled agarose beads (Sigma), prewashed three times with binding buffer (24), were incubated for 60 min at 4°C with 5 g of purified wild-type MP (wtMP; prepared as described previously [3]). The beads were then pelleted by centrifugation (5 min at 18,000 ϫ g) and washed three times with binding buffer to remove unbound proteins. Pellet (bound protein) and supernatant (unbound protein) fractions were analyzed by immunoblotting with anti-MP antibodies (15), which showed that MP binds to the GTP-coupled beads (Fig. 1A). When the MP was incubated for 60 min at 4°C with 2.5 mM GTP prior to incubation with the GTP-agarose beads, most of the MP remained in the supernatant fraction, showing that the binding of MP is to GTP and not directly to the agarose beads (Fig. 1B). Furthermore, preincubation with 2.5 mM ATP, CTP, or UTP did not prevent the binding of MP to the GTP-agarose beads (Fig. 1C), indicating that the CPMV MP specifically binds GTP, like the MPs of TMV and Cucumber mosaic virus (19).In CPMV MP mutant AM5 (1), V142 and D143 (the D motif), which are located in the putative rNTP-...

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The C-terminal region of the movement protein of Cowpea mosaic virus is involved in binding to the large but not to the small coat protein

Cited by 50 publications

References 23 publications

Identification of a Movement Protein of Rice Yellow Stunt Rhabdovirus

Identification of a Movement Protein of Rice Yellow Stunt Rhabdovirus

Studies on the origin and structure of tubules made by the movement protein of Cowpea mosaic virus

The Movement Protein of Cowpea Mosaic Virus Binds GTP and Single-Stranded Nucleic Acid In Vitro

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